Intra-tumoral heterogeneity confounds molecular taxonomy, fuels metastasis, and increases the chances of treatment failure. Understanding the origins of intra-tumoral heterogeneity and developing effective countermeasures should improve cancer outcomes. This proposal focuses on the cellular and molecular origins of intra-tumoral heterogeneity in basal-like breast cancer (BLBC), as these cancers frequently resist chemotherapy and currently lack molecular targets for drug development. BLBC is distinguishable from other breast cancer subtypes, as it exhibits a gene expression signature that is associated with fetal mammary stem cells (fMaSCs) generated during embryogenesis. Importantly, fMaSC-like cancer cells are very tumorigenic and differ significantly from the breast cancer stem cells that have received much recent attention. This project will determine the molecular programs that drive embryonic mammary cells into the stem cell state, and use gene editing technologies to generate a new mouse model that will enable the lab to identify fMaSCs in real time based on the cytokeratins they express. This experimental approach will allow the lab to selectively eliminate these cells to determine unambiguously if these are the only stem cells within the mammary gland, and whether other cells can acquire stemness in response to wounding, inflammation, obesity, etc. p53 mutations are frequently found in BLBC and contribute to both genetic heterogeneity and increased reprogramming efficiency. This project will induce p53 mutations in fMaSCs or their differentiated progeny and ask whether different types of tumors arise, and assess cellular and molecular heterogeneity. This project will determine whether other BLBC relevant mutations, such as BRCA1 (alone or in combination with p53 mutations) or environmental challenges (such as inflammation or obesity) elicit the same effects. Gene expression signatures of resulting tumors will be compared to those of human BLBC to generate mouse models that reflect the human disease more faithfully. Finally, this project will apply single cell sequencing and sophisticated bioinformatic approaches to: 1) decipher the mechanisms by which the stem cell state is generated, 2) assess heterogeneity within the tumor cell population, and 3) determine whether fMaSC embryonic antigens are detectable in human BLBC. Such antigens, and the pathways discovered to drive the fMaSC state, will provide new targets for developing tumor-selective, immune- and molecularly targeted therapies. The fMaSC-like cells in BLBC resemble bona-fide multi-potent embryonic mammary stem cells, and comprise a new and understudied cell type in cancer. Cells with similar stem-like attributes have been described in diverse solid tumors, indicating that findings from these studies will likely have general relevance for cancer biology.